CN117487816B - Application of ZNF709 gene in the preparation of drugs for treating PBC - Google Patents

Abstract

The present invention relates to the field of biomedicine, and in particular to the application of the ZNF709 gene in the preparation of a drug for treating PBC. The sequence of the ZNF709 gene is shown in SEQ ID NO.1, and the PBC is primary biliary cholangitis. The research results of the present invention show that the pathogenesis of primary biliary cholangitis is closely related to the increased formation of neutrophil extracellular traps (NETs) and the induction of PBC-like changes in normal bile duct epithelial cells. The present invention also provides a new target for the treatment of PBC. After overexpressing the ZNF709 gene, the activation of the P53-MDM2 pathway caused by NETs can be partially offset, and the PBC-like changes in bile duct epithelial cells induced by NETs can be partially alleviated. This result provides an important scientific basis for further diagnosis and treatment of PBC and monitoring the progression of PBC.

Description

Application of ZNF709 gene in the preparation of drugs for treating PBC

Technical Field

The present invention relates to the field of biomedicine, and in particular to application of ZNF709 gene in preparing medicine for treating PBC.

Background technique

Primary biliary cholangitis (PBC) is a chronic liver disease that mainly invades bile duct epithelial cells, leading to cholestasis, liver inflammation and fibrosis. The exact pathogenesis of PBC is still not fully understood, which leads to challenges in treatment and management. Traditional treatment of PBC mainly uses ursodeoxycholic acid (UDCA) for drug treatment. However, due to the different responsiveness of patients to UDCA and the limited efficacy of UDCA, some PBC patients have rapid disease progression and may require liver transplantation in the late stage. Neutrophil extracellular traps (NETs) are DNA-protein complexes released by neutrophils. They play an important role in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid antibody syndrome, granulomatosis with polyangiitis and Sjögren's syndrome, but their association with PBC is still unclear. Finding the key genes that promote the occurrence and progression of PBC by NETs can provide an important basis for the diagnosis, individualized treatment and disease monitoring of PBC.

Summary of the invention

In view of the defects and shortcomings of the existing technology, the present invention aims to analyze the connection between NETs and PBC, discover the key targets and mechanisms of action of NETs in promoting the pathogenesis of PBC, and is expected to discover a new therapeutic target, providing a new treatment option for patients who do not respond well to ursodeoxycholic acid treatment, and providing a new target for the diagnosis and disease monitoring of PBC patients.

The first aspect of the present invention is to provide the use of ZNF709 gene in preparing a drug for treating PBC, wherein the sequence of the ZNF709 gene is shown in SEQ ID NO.1.

Furthermore, the drug contains the ZNF709 gene or ZNF709 protein.

Furthermore, the drug also includes a pharmaceutically acceptable carrier.

Furthermore, primary biliary cholangitis is associated with increased neutrophil extracellular trap formation.

The second aspect of the present invention is to provide a recombinant vector comprising the ZNF709 gene.

The third aspect of the present invention is to provide a recombinant lentivirus comprising the recombinant vector.

The fourth aspect of the present invention is to provide stem cells transfected with the recombinant vector or the recombinant lentivirus.

The stem cells are hematopoietic stem cells or mesenchymal stem cells. The hematopoietic stem cells are derived from bone marrow, peripheral blood or umbilical cord blood, and the mesenchymal stem cells are derived from bone marrow, skin, fat, placenta, umbilical cord, umbilical cord blood or menstrual blood.

The fifth aspect of the present invention is to provide the use of the recombinant vector, the recombinant lentivirus or the stem cell in the preparation of a drug for treating PBC.

The sixth aspect of the present invention is to provide a drug for treating PBC, wherein the drug comprises the ZNF709 gene, the recombinant vector, the recombinant lentivirus or the stem cell.

Furthermore, the drug also includes a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier includes a conventional diluent, such as water for injection.

The present invention has the following beneficial effects:

The experimental results of the present invention reveal for the first time the key role of NETs in the pathogenesis of PBC, including that NETs induce PBC-like changes in bile duct epithelial cells by regulating the ZNF709-P53-MDM2 pathway. The present invention has discovered a new mechanism for the pathogenesis of PBC, and ZNF709 is a new target for the treatment of PBC. The results provide an important scientific basis for further diagnosis and treatment of PBC and monitoring the progression of PBC.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the role of NETs in the pathogenesis of PBC, including:

A is a representative immunofluorescence staining image of NETs in peripheral blood neutrophils of PBC patients;

B is a scatter plot of relative quantitative analysis of free DNA in the serum of PBC patients;

C is a representative immunofluorescence staining image of NETs in the liver of PBC patients;

D is a representative immunofluorescence staining image of NETs in the liver of PBC model mice.

Figure 2 shows that NETs downregulate ZNF709 expression, activate the P53-MDM2 pathway, and induce PBC-like changes in bile duct cells, where:

A is a volcano plot of differential gene expression in bile duct epithelial cells after NETs stimulation;

B is a protein immunoblot of the expression levels of ZNF709-P53-MDM2 pathway proteins in bile duct epithelial cells after NETs stimulation;

C is a protein immunoblot of the expression levels of P53-MDM2 pathway proteins in bile duct epithelial cells after knockdown of ZNF709;

D is a western blot of the protein expression levels of ZO-1 and CK-7 in bile duct epithelial cells after knockdown of ZNF709;

E is a protein immunoblot of the protein expression levels of α-SMA, TGF-β, and IL-8 in bile duct epithelial cells after knockdown of ZNF709;

F is a western blot of the N-cadherin protein expression level in bile duct epithelial cells after knockdown of ZNF709;

G is a western blot of the H2AX protein expression level in bile duct epithelial cells after knockdown of ZNF709;

H is a western blot of the PDC-E2 protein expression level in bile duct epithelial cells after knockdown of ZNF709;

I is a scatter plot of the apoptosis level of bile duct epithelial cells detected by flow cytometry after knockdown of ZNF709.

FIG3 shows that overexpression of ZNF709 can partially alleviate PBC-like changes in bile duct epithelial cells induced by NETs, where:

A is a protein immunoblot of the expression levels of P53-MDM2 pathway proteins in bile duct epithelial cells after overexpression of ZNF709;

B is a western blot of the protein expression levels of ZO-1 and CK-7 in bile duct epithelial cells after overexpression of ZNF709;

C is a western blot of the protein expression levels of α-SMA, TGF-β, and IL-8 in bile duct epithelial cells after overexpression of ZNF709;

D is a western blot of the N-cadherin protein expression level in bile duct epithelial cells after overexpression of ZNF709;

E is a western blot of the H2AX protein expression level in bile duct epithelial cells after overexpression of ZNF709;

F is a western blot of the expression level of PDC-E2 protein in bile duct epithelial cells after overexpression of ZNF709;

G is a scatter plot of the apoptosis level of bile duct epithelial cells detected by flow cytometry after overexpression of ZNF709.

Detailed ways

The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

The following example of the present invention discloses the use of the ZNF709 gene in preparing a drug for treating PBC. The sequence of the ZNF709 gene is shown in SEQ ID NO.1.

Example 1: Relationship between NETs and PBC pathogenesis

1 Experimental methods

Peripheral blood neutrophils were extracted from PBC patients and healthy controls, and the level of NETs released by peripheral blood neutrophils was detected by immunofluorescence co-staining of SytoxGreen and MPO-Cy3. Liver tissues of PBC patients and healthy controls were frozen and sectioned, and the level of NETs in liver tissues was observed by immunofluorescence co-staining of CitH3-FITC and MPO-Cy3. Free DNA in serum of PBC patients and healthy controls was semi-quantitatively analyzed by SytoxGreen fluorescence. Liver tissues of PBC model mice and wild C57/BL6 mice were frozen and sectioned, and the level of NETs in liver tissues was observed by immunofluorescence co-staining of CitH3-FITC and MPO-Cy3.

2 Experimental results

Through a series of experiments and studies, this example first discovered that NETs occurred significantly in PBC patients (Figure 1A-C) and in the PBC animal model induced by 2OA-BSA combined with Poly I: C (Figure 1D). The results show that NETs are closely related to the onset of PBC.

Example 2: Expression of ZNF709 gene in bile duct cells

1 Experimental methods

Peripheral blood was collected from healthy subjects by venipuncture and neutrophils were isolated. The isolated neutrophils were resuspended in RPMI-1640 medium in a 24-well plate (0.5×10 ⁶ /well) and stimulated with 1nM phorbol ester (PMA) for 4 hours at 37°C and 5% CO ₂ to generate NETs. After 4 hours, NETs were detected by shaking a microplate at 500 rpm for 5 minutes. The supernatant was collected, and cells and cell debris were filtered with a 0.45μm PES membrane filter. NETs were obtained after washing with PBS three times.

Biliary epithelial cells were seeded in 6-well plates (2.5×10 ⁶ /well), and NETs induced by the above method were co-cultured with bile duct epithelial cells for 48 h. Total RNA of bile duct epithelial cells was collected, and RNA sequencing was used to analyze the effect of NETs on the transcriptome of normal human bile duct epithelial cells.

2 Experimental results

ZNF709 was downregulated in cholangiocytes after NETs treatment ( Figure 2A ).

Example 3: Role of the ZNF709-P53-MDM2 pathway in the development of NETs-induced PBC

1 Experimental methods

Western blot was used to detect the changes of various proteins in the ZNF709-P53-MDM2 pathway after NETs stimulated bile duct epithelial cells, and GAPDH was used as an internal reference.

2 Experimental results

Under the action of NETs, the expression of ZNF709 in bile duct epithelial cells was downregulated, the expression of P53 and MDM2 was upregulated, and the P53-MDM2 pathway was activated ( Figure 2B ).

Example 4: Effect of knocking down the ZNF709 gene on bile duct epithelial cells

1 Experimental methods

(1) siRNA (sequence shown in Table 1) was transfected into bile duct epithelial cells to knock down the expression level of ZNF709. When the cells grew to 80-90%, total protein was extracted by RIPA. The expression levels of P53 and MDM2 in bile duct epithelial cells were detected by Western blot, with GAPDH as an internal reference.

Table 1 siRNA sequences

(2) Biliary epithelial cells were transfected with siRNA to knock down the expression level of ZNF709. When the cells grew to 80-90%, total protein was extracted using RIPA. The expression levels of ZO-1, CK-7, α-SMA, TGF-β, IL-8, N-cadherin, H2AX, and PDC-E2 proteins in bile duct epithelial cells were detected by Western blot, with GAPDH as an internal reference.

(3) siRNA was transfected into bile duct epithelial cells to knock down the expression level of ZNF709. When the cells grew to 80-90%, the level of cell apoptosis (Annexin V-FITC, PI) was detected by flow cytometry.

2 Experimental results

(1) After knockdown of ZNF709, the P53-MDM2 pathway was activated ( Figure 2C ).

(2) After knockdown of ZNF709, bile duct epithelial cells underwent epithelial changes (Figure 2D), inflammation and fibrosis (Figure 2E), EMT (Figure 2F), DNA damage (Figure 2G), and overexpression of the bile duct epithelial cell PBC-specific antigen PDC-E2 (Figure 2H).

(3) After knockdown of ZNF709, bile duct epithelial cells underwent apoptosis ( Figure 2I ).

Example 5: Effect of overexpression of ZNF709 gene on bile duct epithelial cells

1 Experimental methods

Lentivirus construction: The ZNF709 gene shown in SEQ ID NO.1 was connected to the BamH I/EcoR I site of the pCDH-CMV-MCS-EF1-Puro vector (Aiji Biotechnology), and the above vector was transfected into HEK293T cells to obtain virus liquid. Biliary epithelial cells were transfected with lentivirus to overexpress the ZNF709 gene. NETs were co-cultured with bile duct epithelial cells for 48 hours. Under the stimulation of NETs, when the cells grew to 80-90%, total protein was extracted with RIPA, and the expression levels of P53-MDM2 pathway proteins in bile duct epithelial cells were detected by Western blot, and the expression levels of ZO-1, CK-7, α-SMA, TGF-β, IL-8, N-cadherin, H2AX, and PDC-E2 proteins were detected, and GAPDH was used as an internal reference. The level of cell apoptosis (Annexin V-FITC, PI) was detected by flow cytometry.

2 Experimental results

Overexpression of the ZNF709 gene could partially offset the activation of the P53-MDM2 pathway caused by NETs (Figure 3A), and could partially alleviate the PBC-like changes of biliary epithelial cells induced by NETs, such as epithelial changes (Figure 3B), inflammation and fibrosis changes (Figure 3C), EMT changes (Figure 3D), DNA damage (Figure 3E), overexpression of the PBC-specific antigen PDC-E2 in biliary epithelial cells (Figure 3F), and cell apoptosis (Figure 3G).

Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (4)

1. The application of ZNF709 gene in preparing medicament for treating PBC is characterized in that the sequence of ZNF709 gene is shown in SEQ ID NO.1, and PBC is primary cholangitis.
2. 2. The use according to claim 1, wherein the medicament comprises the ZNF709 gene or a protein encoded by the ZNF709 gene.
3. 3. The use of claim 2, wherein the medicament further comprises a pharmaceutically acceptable carrier.
4. 4. Use of a recombinant vector comprising the ZNF709 gene of claim 1 or a recombinant lentivirus comprising the recombinant vector for the preparation of a medicament for treating PBC, wherein the sequence of the ZNF709 gene is shown in SEQ ID No.1, and the PBC is primary cholangitis.